Abstract:
Objective To establish Saposhnikovia divaricata (Turcz.) Schischk. hairy root culture system overexpressing p-coumaroyl CoA 2'-hydroxylase (C2'H) gene, and explore the regulatory role of SdC2'H gene in coumarin biosynthesis.
Method The SdC2'H gene was cloned. A plant binary expression vector overexpressing SdC2'H was constructed through gene fusion method. The S. divaricata hairy roots were induced by using Agrobacterium rhizogenes K599 transformation method. The expression level of SdC2'H gene was analyzed by using RT-qPCR. The coumarin content in the hairy roots of S. divaricata was determined by HPLC.
Result The open reading frame of S. divaricata SdC2'H gene (GenBank accession number: PV169361) was 813 bp in length, and the gene encoded a 270-amino acid protein. The SdC2'H protein was a hydrophilic, unstable protein with no signal peptide and was located outside the membrane. The phylogenetic tree showed that the SdC2'H protein had the closest relationship with Angelica decursiva. In S. divaricata hairy roots overexpressing the SdC2'H gene, the expression level of SdC2'H was significantly elevated compared to blank control (P<0.0001), while the coumarin content markedly exceeded both the blank control (P<0.001) and the empty vector negative control (P<0.01). This indicated that overexpressing SdC2'H gene could improve coumarin content in hairy roots of S. divaricata.
Conclusion This study confirms that SdC2'H gene can promote coumarin biosynthesis in S. divaricata, provides a theoretical basis for analyzing the role of SdC2'H gene in metabolism of coumarins, and lays a foundation for investigating molecular regulatory network of coumarin biosynthesis.
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